Goat anti collagen 3

Protein lysates were prepared by mechanical shearing of frozen bladders by pestle and mortar, followed by protein extraction for 30 min at 4 °C in cold RIPA buffer. Lysates were centrifuged, and supernatant was collected and stored at −80 °C. Samples were diluted to 30 ng/L and heated at 95 °C for 5 min in 4× loading buffer. Western blots were performed on 7% (for collagen I and III) or 10% (for alpha-smooth muscle actin) SDS gels run under reducing conditions for 1 h. Protein was then transferred to PVDF membranes at 100 V for 1.5 h. Membranes were washed in PBS and blocked in 3% BSA before incubating with primary antibodies for goat anti-Collagen I (Southern Biotech), goat anti- Collagen III (Southern Biotech, Birmingham, AL, USA) 1/1000, a-SMA (Abcam) 1/5000, and GAPDH (Invitrogen, Waltham, MA, USA) 1/10,000 overnight at 4 °C. Membranes were then washed and incubated with an HRP-conjugated secondary antibody (Santa Cruz, Dallas, TX, USA) 1/5000 for 1 h. Membranes were washed and exposed to peroxide substrate for 5 min before imaging using the Amersham Imager 600. Quantification was done using Fiji.

Immunohistochemical (IHC) staining of collagen III was performed to quantitate renal fibrosis. Paraffin-embedded sections were deparaffinised and hydrated followed by antigen retrieval with proteinase K (10 μg/ml) treatment for 10 minutes at 37°C and treated in TBS for 5 minutes. Endogenous peroxidase was blocked using 0.5% H₂O₂ in TBS for 20 minutes, and sections were incubated with avidin and biotin for 10 min each. Sections were incubated with TBS mixed with 7% donkey, 3% mouse serum, and 3% skimmed milk. Subsequently, sections were incubated overnight at 4°C with goat anticollagen III (Southern Biotech, Birmingham, USA) diluted in 7% donkey, 3% mouse serum, and 0.5% skimmed milk in TBS. The next day, the sections were incubated in biotinylated donkey anti-goat IgG (cat. number 705-065-147) diluted in 7% donkey, 3% mouse serum, and 0.5% skimmed milk in TBS and afterwards in vectastain ABC complex in TBS. Specific binding of antibodies was visualised by enzymatic conversion of the chromogenic substrate DAB into a brown precipitate by HRP activated by hydrogen peroxide. The slides were counterstained with haematoxylin.